Glucose dehydrogenase and method for producing the dehydrogenase

ABSTRACT

A glucose dehydrogenase having high substrate specificity can be produced at a low cost by culturing a microorganism belonging to the genus  Burkholderia . The glucose dehydrogenase is isolated from the medium and/or cells of the microorganism. The glucose dehydrogenase isolated from the genus  Burkholderia  is not affected by oxygen dissolved in a measurement sample and, in particular, has superior thermal stability.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/415,504, filed Jul. 3, 2003, now U.S. Pat. No. 7,741,090, which isincorporated herein by reference and which is the U.S. National Phaseunder 35 U.S.C. §371 of International Application PCT/JP01/09556, filedOct. 31, 2001, which was published in the Japanese language which claimsthe priority of Japanese Application No. 2001-276832, filed Sep. 12,2001; Japanese Application No. 2000-357102, filed Nov. 24, 2000; andJapanese Application No. 2000-332085, filed Oct. 31, 2000.

TECHNICAL FIELD

The present invention relates to a novel glucose dehydrogenase and amethod for producing the same, a DNA encoding the enzyme, a recombinantvector comprising the DNA encoding the enzyme, a transformanttransformed with the recombinant vector, a novel microorganism producingthe enzyme, a glucose sensor using an enzyme electrode including theenzyme, the transformant or the microorganism, and a glucose assay kit.

BACKGROUND ART

Biosensors using an enzyme that specifically reacts with a particularsubstrate are being actively developed in various industrial fields. Asfor a glucose sensor, which is one of the biosensors, in particular,measurement methods and devices utilizing such methods are beingactively developed mainly in medical fields.

The glucose sensor has a history of about 40 years since Clark and Lyonsfirst reported about a biosensor comprising glucose oxidase and anoxygen electrode in combination in 1962 (L. c. Clark, J. and Lyonas, C.“Electrode systems for continuous monitoring in cardiovascular surgery.”Ann. n.y. Acad. Sci., 105: 20-45).

Thus, the adoption of glucose oxidase as an enzyme of the glucose sensorhas a long history. This is because glucose oxidase shows high substratespecificity for glucose and superior thermal stability, this enzyme canfurther be produced in a large scale, and its production cost is lowerthan those of other enzymes.

The high substrate specificity means that this enzyme does not reactwith a saccharide other than glucose, and this leads to an advantagethat accurate measurement can be achieved without error in measurementvalues.

Further, the superior thermal stability means that problems concerningdenaturation of the enzyme and inactivation of its enzymatic activitydue to heat can be prevented, and this leads to an advantage thataccurate measurement can be performed over a long period of time.

However, although glucose oxidase has high substrate specificity andsuperior thermal stability and can be produced at a low cost, it has aproblem that the enzyme is affected by dissolved oxygen as describedbelow and this affects measurement results.

Meanwhile, in addition to glucose oxidase, a glucose sensor utilizingglucose dehydrogenase has also been developed. This enzyme is also foundin microorganisms.

For example, there are known glucose dehydrogenase derived from Bacillusbacteria (EC 1.1.1.47) and glucose dehydrogenase derived fromCryptococcus bacteria (EC 1.1.1.119).

The former glucose dehydrogenase (EC 1.1.1.47) is an enzyme thatcatalyzes a reaction ofβ-D-glucose+NAD(P)⁺>D-δ-gluconolactone+NAD(P)H+H⁺, and the latterglucose dehydrogenase (EC1.1.1.119) is an enzyme that catalyzes areaction of D-glucose+NADP⁺>D-δ-gluconolactone+NADPH+H⁺. Theaforementioned glucose dehydrogenases derived from microorganisms arealready marketed.

These glucose dehydrogenases have an advantage that they are notaffected by oxygen dissolved in a measurement sample. This leads to anadvantage that accurate measurement can be achieved without causingerrors in measurement results even when the measurement is performed inan environment in which the oxygen partial pressure is low, or ahigh-concentration sample requiring a large amount of oxygen is used forthe measurement.

However, although glucose dehydrogenase is not affected by dissolvedoxygen, it has problems of poor thermal stability and substratespecificity poorer than that of glucose oxidase.

Therefore, an enzyme that overcomes disadvantages of both of glucoseoxidase and glucose dehydrogenase has been desired.

The inventors of the present invention reported results of their studiesabout glucose dehydrogenase using samples collected from soil near hotsprings in Sode K., Tsugawa W., Yamazaki T., Watanabe M., Ogasawara N.,and Tanaka M., Enzyme Microb. Technol., 19, 82-85 (1996); Yamazaki T.,Tsugawa W. and Sode K., Appli. Biochem. and Biotec., 77-79/0325 (1999);and Yamazaki T., Tsugawa W. and Sode K., Biotec. Lett., 21, 199-202(1999).

However, a bacterial strain having oxygen-producing ability had not beenidentified at the stage of these studies.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an enzyme thatovercomes the disadvantages of both of known glucose oxidase and glucosedehydrogenase, i.e., an enzyme that shows high substrate specificity andsuperior thermal stability, can be produced at a low cost and is notaffected by oxygen dissolved in a measurement sample.

Further, another object of the present invention is to provide a methodfor producing the aforementioned enzyme, a protein utilizingcharacteristics of the enzyme and a novel microorganism producing theenzyme.

A further object of the present invention is to provide a DNA encodingthe aforementioned enzyme, a recombinant vector containing the DNAencoding the enzyme and a transformant transformed with the recombinantvector.

A still further object of the present invention is to provide a glucosesensor using an enzyme electrode including the aforementioned enzyme,transformant or microorganism and a glucose assay kit including theaforementioned enzyme.

The inventors of the present invention successfully isolatedBurkhorderia cepacia producing an enzyme achieving the aforementionedobjects from soil near hot springs, and thus accomplished the presentinvention.

Thus, the present invention provides the followings.

(1) A method for producing glucose dehydrogenase comprising the steps ofculturing a microorganism belonging to the genus Burkhorderia and havingglucose dehydrogenase producing ability in a medium, and collectingglucose dehydrogenase from the medium and/or cells of the microorganism.(2) The method for producing glucose dehydrogenase according to (1),wherein the microorganism is Burkhorderia cepacia.(3) The method for producing glucose dehydrogenase according to (1) or(2), wherein the glucose dehydrogenase has the following properties:(i) the enzyme has an action of catalyzing dehydrogenation reaction ofglucose;(ii) the enzyme consists of subunits showing a molecular weight of about60 kDa and a molecular weight of about 43 kDa in SDS-polyacrylamide gelelectrophoresis under a reducing condition;(iii) the enzyme shows a molecular weight of about 380 kDa in gelfiltration chromatography using TSK Gel G3000SW (Tosoh Corporation); and(iv) the enzyme shows an optimal reaction temperature around 45° C.(Tris-HCl buffer, pH 8.0).(4) The method for producing glucose dehydrogenase according to (3),wherein the subunit showing a molecular weight of about 43 kDa is anelectron-transferring protein.(5) The method for producing glucose dehydrogenase according to (4),wherein the electron-transferring protein is cytochrome C.(6) A glucose dehydrogenase, which can be produced by a microorganismbelonging to the genus Burkhorderia.(7) The glucose dehydrogenase according to (6), wherein themicroorganism is Burkhorderia cepacia.(8) The glucose dehydrogenase according to (6) or (7), wherein theglucose dehydrogenase has the following properties:(i) the enzyme has an action of catalyzing dehydrogenation reaction ofglucose;(ii) the enzyme consists of subunits showing a molecular weight of about60 kDa and a molecular weight of about 43 kDa in SDS-polyacrylamide gelelectrophoresis under a reducing condition;(iii) the enzyme shows a molecular weight of about 380 kDa in gelfiltration chromatography using TSK Gel G3000SW (Tosoh Corporation); and(iv) the enzyme shows an optimal reaction temperature around 45° C.(Tris-HCl buffer, pH 8.0).(9) The glucose dehydrogenase according to (8), wherein the subunitshowing a molecular weight of about 43 kDa is an electron-transferringprotein.(10) The glucose dehydrogenase according to (9), wherein theelectron-transferring protein is cytochrome C.(11) The glucose dehydrogenase according to any one of (8) to (10),wherein the subunit showing a molecular weight of about 60 kDa comprisesthe amino acid sequence of the amino acid numbers 2 to 12 in SEQ ID NO:3.(12) The glucose dehydrogenase according to any one of (8) to (11),wherein the N-terminus of the subunit showing a molecular weight of 43kDa has the amino acid sequence of SEQ ID NO: 5.(13) The glucose dehydrogenase according to (11), wherein the subunitshowing a molecular weight of about 60 kDa is a protein defined in thefollowing (A) or (B):(A) a protein which has the amino acid sequence of SEQ ID NO: 3;(B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.(14) The glucose dehydrogenase according to (6), which shows activitypeaks around 45° C. and around 75° C.(15) A cytochrome C, which is a subunit of the glucose dehydrogenaseaccording to (10) and has the amino acid sequence of SEQ ID NO: 5.(16) A DNA encoding a part of the cytochrome C according to (15) andhaving the nucleotide sequence of SEQ ID NO: 8.(17) A DNA encoding a part of the cytochrome C according to (15) andhaving the nucleotide sequence of the nucleotide numbers 2386 to 2467 inthe nucleotide sequence of SEQ ID NO: 1.(18) A DNA encoding a signal peptide of the cytochrome C according to(15) and comprising the nucleotide sequence of the nucleotide numbers2386 to 2451 in the nucleotide sequence of SEQ ID NO: 1.(19) A peptide which is a signal peptide of cytochrome C and has theamino acid sequence of the amino acid numbers 1 to 22 in the amino acidsequence of SEQ ID NO: 4.(20) A protein having the following properties:(i) the protein can constitute the glucose dehydrogenase according to(6) as a subunit;(ii) the protein has a glucose dehydrogenase activity;(iii) the protein shows a molecular weight of about 60 kDa inSDS-polyacrylamide gel electrophoresis under a reducing condition; and(iv) the protein shows an optimal reaction temperature around 75° C.(Tris-HCl buffer, pH 8.0).(21) The protein according to (20), which comprises the amino acidsequence of the amino acid numbers 2 to 12 in SEQ ID NO: 3.(22) The glucose dehydrogenase according to (21), wherein the protein isa protein defined in the following (A) or (B) defined in the following(A) or (B):(A) a protein which has the amino acid sequence of SEQ ID NO: 3;(B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.(23) A protein defined in the following (A) or (B):(A) a protein which has the amino acid sequence of SEQ ID NO: 3;(B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.(24) A DNA encoding a protein defined in the following (A) or (B):(A) a protein which has the amino acid sequence of SEQ ID NO: 3;(B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.(25) The DNA according to (24), which is a DNA defined in the following(a) or (b):(a) a DNA which comprises the nucleotide sequence of the nucleotidenumbers 764 to 2380 in the nucleotide sequence of SEQ ID NO: 1;(b) a DNA which is hybridizable with a nucleotide sequence comprisingthe sequence of the nucleotide numbers 764 to 2380 in SEQ ID NO: 1 or aprobe that can be prepared from the sequence under a stringent conditionand encodes a protein having a glucose dehydrogenase activity.(26) A recombinant vector comprising the DNA according to (24) or (25).(27) The recombinant vector according to (26), which comprisesnucleotide sequences encoding the signal peptide according to (18) and aβ-subunit.(28) A transformant transformed with the DNA according to (24) or (25)or the recombinant vector according to (26) or (27).(29) A method for producing glucose dehydrogenase comprising the stepsof culturing the transformant according to (28) to produce glucosedehydrogenase as an expression product of the DNA, and collecting it.(30) A Burkhorderia cepacia KS1 strain (FERM BP-7306).(31) A glucose sensor using an enzyme electrode including the glucosedehydrogenase according to any one of (6) to (14), the protein accordingto any one of (20) to (23), the transformant according to (27) or thestrain according to (30).(32) A glucose assay kit including the glucose dehydrogenase accordingto any one of (6) to (14) or the protein according to any one of (20) to(23).(33) A protein having the amino acid sequence of SEQ ID NO: 2.(34) A DNA encoding a protein having the amino acid sequence of SEQ IDNO: 2.(35) The DNA according to (34), which comprises the nucleotide sequenceof the nucleotide numbers 258 to 761 in the nucleotide sequence of SEQID NO: 1.(36) A DNA comprising the DNA according to (34) or (35) and the DNAaccording to (24) or (25) in this order.(37) The DNA according to (36), which comprises the nucleotide sequenceof the nucleotide numbers 258 to 2380 in the nucleotide sequence of SEQID NO: 1.(38) A recombinant vector comprising the DNA according to (36) or (37).(39) The recombinant vector according to (38), which comprisesnucleotide sequences encoding the signal peptide according to (18) and aβ-subunit.(40) A transformant transformed with the DNA according to (36) or (37)or the recombinant vector according to (38) or (39).(41) A method for producing glucose dehydrogenase comprising the stepsof culturing the transformant according to (40) to produce glucosedehydrogenase as an expression substance of the DNA according to (36) or(37), and collecting it.

Hereafter, the present invention will be explained in detail.

<1> Novel Bacterial Strain Producing Glucose Dehydrogenase of thePresent Invention

The enzyme of the present invention (hereinafter, also referred to as“the enzyme” or “GDH”) can be produced by a bacterium belonging to thegenus Burkhorderia. The Burkhorderia bacterium used for the presentinvention is not particularly limited so long as it is a Burkhorderiabacterium having ability to produce the enzyme. However, Burkhorderiacepacia, in particular, the Burkhorderia cepacia KS1 strain ispreferred. This bacterial strain is a novel bacterial strain isolated bythe inventors of the present invention from soil near hot springs asdescribed later in the examples and was identified as Burkhorderiacepacia based on its bacteriological properties. Conventionally, it hasbeen unknown that a microorganism belonging to the genus Burkhorderiacan produce glucose dehydrogenase. This bacterial strain was designatedas KS1 strain. This strain was deposited at International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, postal code: 305-8566) on Sep. 25, 2000 and receiveda microorganism accession number of FERM BP-7306.

The inventors of the present invention obtained some Burkhorderiacepacia strains other than the Burkhorderia cepacia KS1 strain, whichwere deposited at Institute for Fermentation (Osaka, IFO) or JapanCollection of Microorganisms (JCM), the Institute of Physical andChemical Research, and measured their glucose dehydrogenase activities.As a result, they confirmed that all of these bacterial strains had theactivity.

<2> Glucose Dehydrogenase of the Present Invention

If a Burkhorderia bacterium having glucose dehydrogenase producingability, for example, the Burkhorderia cepacia KS1 strain, is culturedin a nutrient medium used for usual culture of a microorganism,preferably a medium containing glucose or a substance containing glucosein order to increase the enzyme producing ability, the glucosedehydrogenase of the present invention is produced and accumulated in aculture product or cultured cells. Therefore, it can be collected by aknown method. The method for producing the enzyme will be specificallyexplained by exemplifying the Burkhorderia cepacia KS1 strain. First,the Burkhorderia cepacia KS1 strain is cultured in a suitable nutrientmedium, for example, a medium containing suitable carbon source,nitrogen source, inorganic salts, glucose or substances containing theseand so forth to produce and accumulate the enzyme in the culture productor the cultured cells.

As the carbon sources, any substance that can be assimilated can beused, and examples include, for example, D-glucose, L-arabinose,D-xylose, D-mannose, starch, various peptones and so forth. As thenitrogen sources, there can be used yeast extract, malt extract, variouspeptones, various meat extracts, corn steep liquor, amino acid solutionsand organic and inorganic nitrogen compounds such as ammonium salts orsubstances containing these. As the inorganic salts, there can be usedvarious phosphoric acid salts and salts of magnesium, potassium, sodium,calcium and so forth. Further, as required, various inorganic andorganic substances required for growth of the bacterium or production ofthe enzyme, for example, silicone oil, sesame oil, defoaming agents suchas various surfactants and vitamins can be added to the medium.

As for the culture method, although either liquid culture or solidculture may be used, liquid culture is usually preferred.

The enzyme of the present invention can be obtained from the mediumand/or the cells in the culture obtained as described above. The enzymeexisting in the cells can be obtained as a cell extract by disrupting orlysing the cells.

The glucose dehydrogenase in the culture product or the cell extract canbe purified by a suitable combination of chromatography techniques usingan ion exchanger, a gel filtration carrier, a hydrophobic carrier and soforth.

The activity of the enzyme can be measured by the same methods as knownmethods for measurement of the glucose dehydrogenase activity.Specifically, the activity can be measured by, for example, the methoddescribed later in the examples.

Physicochemical properties of the novel glucose dehydrogenase of thepresent invention are as follows:

(i) the enzyme has an action of catalyzing dehydrogenation reaction ofglucose;

(ii) the enzyme consists of subunits showing a molecular weight of about60 kDa and a molecular weight of about 43 kDa in SDS-polyacrylamide gelelectrophoresis under a reducing condition;

(iii) the enzyme shows a molecular weight of about 380 kDa in gelfiltration chromatography using TSK Gel G3000SW (Tosoh Corporation); and

(iv) the enzyme shows an optimal reaction temperature around 45° C.(Tris-HCl buffer, pH 8.0).

The glucose dehydrogenase shows an activity peak around 45° C. under theaforementioned condition, and also shows an activity peak around 75° C.(refer to FIG. 3 (a)) No GDH has been known which shows the activitypeak in two of temperature regions as described above.

The molecular weight and the optimal temperature can be measured by themethods described later in the examples.

The aforementioned glucose dehydrogenase of the present inventionconsists of two of separate polypeptides, the α-subunit having amolecular weight of about 60 kDa and the β-subunit having a molecularweight of about 43 kDa (hereinafter, this glucose dehydrogenase is alsoreferred to as “multimer enzyme”). The inventors of the presentinvention further investigated these two of subunits in detail.

It was found that the β-subunit was cytochrome C (as shown later in theexamples). A protein containing only the α-subunit exhibits thefollowing physicochemical properties:

(i) the protein can constitute the glucose dehydrogenase as a subunit;

(ii) the protein has a glucose dehydrogenase activity;

(iii) the protein shows a molecular weight of about 60 kDa inSDS-polyacrylamide gel electrophoresis under a reducing condition; and

(iv) the protein shows an optimal reaction temperature around 75° C.(Tris-HCl buffer, pH 8.0).

The optimal temperature can be measured by the method described later inthe examples.

Since this protein itself has the enzymatic activity, the protein may beoptionally called as “peptide enzyme” or “enzyme” depending on thecontent of the explanation.

As a specific embodiment of the peptide enzyme of the present invention,a protein having the amino acid sequence of SEQ ID NO: 3 can bementioned. Further, this peptide enzyme may be a protein having theamino acid sequence containing substitution, deletion, insertion oraddition of one or more amino acid residues in the amino acid sequenceof SEQ ID NO: 3 so long as it has the GDH activity. Although an aminoacid sequence that can be encoded by the nucleotide sequence of SEQ IDNO: 1 is shown as SEQ ID NO: 3, the methionine residue at the N-terminusmay be eliminated after translation.

Further, as a specific embodiment of the multimer enzyme of the presentinvention, there can be mentioned a multimer containing a protein ofwhich α-subunit has the amino acid sequence of SEQ ID NO: 3. Further,the aforementioned multimer enzyme may be a multimer containing aprotein of which α-subunit has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one or moreamino acid residues, so long as it has the GDH activity.

In the present invention, “one or more” means a number of 1 to 10,preferably 1 to 5, particularly preferably 1 to 3.

The inventors of the present invention confirmed existence of aγ-subunit in addition to the aforementioned α-subunit and β-subunit.

In the examples described later, the γ-subunit was removed at the stageof purifying the enzyme of the present invention from a culturesupernatant or cell extract, and therefore the γ-subunit was notconfirmed in the purified enzyme. However, as shown in the examples,when the γ-subunit was expressed together with the α-subunit, a highenzymatic activity was obtained in comparison with the case where onlythe α-subunit was expressed. This suggested that the γ-subunit was aprotein involved in the production of the α-subunit in a microbial cellin some sort of way. Assuming that the specific activity of theα-subunit (enzymatic activity per protein) is the same in either case, alower enzymatic activity indicates a smaller amount of the α-subunit asan enzyme since the enzymatic activity reflects the amount of theenzyme. On the other hand, the produced α-subunit may be protected bythe γ-subunit in a certain manner, or although the α-subunit as aprotein is fully expressed, it cannot have the three-dimensionalstructure for exhibiting the enzymatic activity due to the absence ofγ-subunit, and thus the enzymatic activity may become low. In eithercase, a high enzymatic activity can be obtained when the γ-subunit isexpressed together with the α-subunit.

<3>DNA of the Present Invention

The DNA of the present invention can be obtained from a microorganismcontaining the DNA of the present invention, for example, Burkhorderiacepacia. The DNA of the present invention was isolated from chromosomalDNA of Burkhorderia cepacia in the process of accomplishing the presentinvention. However, since its nucleotide sequence and the amino acidsequence encoded by this nucleotide sequence were elucidated by thepresent invention, the DNA can also be obtained by chemical synthesisbased on those sequences. Further, the DNA of the present invention canalso be obtained from chromosomal DNA of Burkhorderia cepacia or thelike by hybridization or PCR using an oligonucleotide prepared based onthe aforementioned sequences as a probe or a primer.

In addition to a DNA which encodes a protein having the amino acidsequence of SEQ ID NO: 3, the DNA of the present invention may be a DNAwhich encodes a protein having an amino acid sequence of SEQ ID NO: 3containing substitution, deletion, insertion or addition of one or moreamino acid residues in the amino acid sequence and has the GDH activity.

As the DNA of the present invention, there can be specifically mentioneda DNA comprising the nucleotide sequence of the nucleotide numbers 764to 2380 in the nucleotide sequence of SEQ ID NO: 1. The nucleotidesequence of the nucleotide numbers 764 to 2380 in the nucleotidesequence of SEQ ID NO: 1 encodes the α-subunit of GDH having the aminoacid sequence of SEQ ID NO: 3.

Further, the DNA of the present invention may also be a DNA which ishybridizable with the nucleotide sequence of the nucleotide numbers 764to 2380 in the nucleotide sequence of SEQ ID NO: 1 or a probe that canbe prepared from the sequence under a stringent conditions and encodes aprotein having the GDH activity.

It is estimated that the nucleotide sequence of the nucleotide numbers258 to 761 in the nucleotide sequence of SEQ ID NO: 1 encodes theγ-subunit. The amino acid sequence is shown in SEQ ID NO: 2. It isconsidered that, since the structural gene of the γ-subunit is includedin a region upstream from that of the α-subunit, and thus the γ-subunitis expressed first and already exists as a protein upon the productionof the α-subunit by a microorganism, the α-subunit can be efficientlyproduced in the microorganism. Therefore, the DNA of the presentinvention may include a DNA encoding the amino acid sequence of SEQ IDNO: 2 in addition to the aforementioned DNA.

A DNA encoding a protein substantially identical to the aforementionedprotein having the amino acid sequence of SEQ ID NO: 3 can be obtainedby, for example, a method such as the site-directed mutagenesis ormutagenesis treatment. The GDH activity of a protein encoded by a DNAintroduced with a mutation can be measured, for example, as follows.

An enzyme sample and glucose as a substrate are added to 10 mM potassiumphosphate buffer (pH 7.0) containing 594 μM methylphenazine methosulfate(mPMS) and 5.94 μM 2,6-dichlorophenol-indopheol (DCIP) and incubated at37° C. Change in absorbance of the DCIP at 600 nm is monitored by usinga spectrophotometer, and the absorbance decreasing rate is measured asan enzymatic reaction rate.

Further, the nucleotide sequence consisting of the nucleotide of thenucleotide number 2386 and the sequence after the nucleotide of thenucleotide number 2386 in the nucleotide sequence of SEQ ID NO: 1 isestimated to encode the β-subunit. Further, the nucleotide sequence ofthe nucleotide numbers 2386 to 2451 is estimated to encode the signalpeptide of the β-subunit. An estimated amino acid sequence of thissignal peptide is the amino acid sequence of amino acid numbers 1 to 22in SEQ ID NO: 4. The signal peptide is a peptide necessary for a proteinsynthesized in ribosome to be secreted through the membrane and has beenfound to comprise 15 to 30 hydrophobic amino acid residues. Therefore,since the amount of proteins in the culture supernatant is increased dueto the existence of the signal peptide, this is a peptide effectivelyacting in a method of producing a protein.

Hereafter, an example of a method for obtaining the DNA of the presentinvention will be explained.

Chromosomal DNA is isolated from a microorganism such as Burkhorderiacepacia and purified, and the chromosomal DNA is cleaved byultrasonication, restriction enzyme treatment or the like and ligated toa linear expression vector and cyclized by using a DNA ligase or thelike to construct a recombinant vector. The obtained recombinant vectoris introduced into a host microorganism in which the vector isautonomously replicable, and the transformants are screened by using avector marker and expression of an enzymatic activity as indexes toobtain a microorganism harboring a recombinant vector containing a geneencoding GDH. The recombinant vector contained in the obtainedmicroorganism is expected to contain at least the nucleotide sequenceencoding the α-subunit. Further, if the cloned fragment has a sufficientsize, it is very likely that the nucleotide sequence encoding theγ-subunit is also contained.

Then, the microorganism having the recombinant vector can be cultured,the recombinant vector can be isolated from the cells of the culturedmicroorganism and purified, and the gene encoding GDH can be collectedfrom the expression vector. For example, chromosomal DNA serving as agene donor is specifically collected, for example, as follows.

The aforementioned gene donor microorganism can be cultured withstirring for 1 to 3 days, for example, and cells can be collected bycentrifugation from the obtained culture broth and then lysed to preparecell lysate containing the GDH gene. As the method for lysis of thecells, a treatment is performed by using a bacteriolytic enzyme such aslysozyme, and other enzymes such as protease and surfactants such assodium dodecylsulfate (SDS) are used in combination as required.Further, a physical cell disruption method such as freeze and thawing orFrench press treatment may also be employed in combination.

The DNA can be isolated and purified from the lysate obtained asdescribed above in a conventional manner, for example, by a suitablecombination of deproteinization by phenol treatment or proteasetreatment, ribonuclease treatment, alcohol precipitation and so forth.

The DNA isolated and purified from a microorganism can be cleaved by,for example, ultrasonication, restriction enzyme treatment or the like.Preferably, a type-II restriction enzyme, which acts on a specificnucleotide sequence, is suitably used. The restriction enzyme used maygenerate an end matching a digested end of a vector, or the digested endmay be blunt-ended by using an arbitrary restriction enzyme and ligatedto the vector.

As the vector used for cloning, a phage that can autonomously grow in ahost microorganism or a plasmid that is constructed for generecombination is suitable. Examples of the phage include, for example,when Escherichia coli is used as the host microorganism, Lambda gt10,Lambda gt11 and so forth. Further, examples of the plasmid include, forexample, when Escherichia coli is used as the host microorganism,pBR322, pUC18, pUC118, pUC19, pUC119, pTrc99A and pBluescript as well asSuperCosI, which is a cosmid, and so forth.

Upon the cloning, a vector fragment can be obtained by digesting theaforementioned vector with a restriction enzyme used for the digestionof a microbial DNA as the aforementioned donor of a gene encoding GDH.However, a restriction enzyme identical to the restriction enzyme usedfor the digestion of the microbial DNA does not necessarily need to beused. The method for ligating the microbial DNA fragment and the vectorDNA fragment may be a known method using a DNA ligase. For example, anadhesive end of the microbial DNA fragment and an adhesive end of thevector fragment are ligated, and then a recombinant vector containingthe microbial DNA fragment and the vector DNA fragment is produced byusing a suitable DNA ligase. If required, after the ligation, thefragment can also be introduced into the host microorganism to producethe recombinant vector by utilizing a DNA ligase existing in themicroorganism.

The host microorganism used for the cloning is not particularly limitedso long as the recombinant vector is stable and can autonomously grow inthe host, and a foreign gene can be expressed in the host. Escherichiacoli DH5α, XL-1 BlueMR and so forth can generally be used.

As the method for introducing the recombinant vector into the hostmicroorganism, for example, when the host microorganism is Escherichiacoli, the competent cell method using calcium treatment, electroporationor the like can be used.

Whether the cloned fragment obtained by the aforementioned methodencodes GDH can be confirmed by decoding the nucleotide sequence of thefragment in a conventional manner.

The DNA of the present invention can be obtained by collecting arecombinant vector from the transformant obtained as described above.

GDH can be produced by culturing a transformant containing the DNA ofthe present invention or a recombinant vector containing the DNA toproduce GDH as an expression product of the DNA and collecting it fromthe cells or culture broth. For this production, although the DNA of thepresent invention may be a DNA encoding the α-subunit, the expressionefficiency can be increased by further expressing the γ-subunit togetherwith the α-subunit.

Examples of the microorganism in which GDH is produced include entericbacteria such as Escherichia coli, Gram-negative bacteria such as thoseof the genera Pseudomonas and Gluconobacter, Gram-positive bacteriaincluding Bacillus bacteria such as Bacillus subtilis, yeasts such asSaccharomyces cerevisiae and filamentous fungi such as Aspergillusniger. However, the microorganism is not limited to thesemicroorganisms, and any host microorganism suitable for production offoreign proteins can be used.

The GDH gene contained in the once selected recombinant vectorcontaining the GDH gene can be easily transferred into a recombinantvector that can be replicated in a microorganism by recovering a DNAwhich is the GDH gene from the recombinant vector containing the GDHgene by using a restriction enzyme or by PCR and ligating it to anothervector fragment. Further, the microorganism can be easily transformedwith these vectors, for example, by the competent cell method usingcalcium treatment for Escherichia bacteria, the protoplast method forBacillus bacteria, the KU or KUR method for yeasts, themicromanipulation method for filamentous fungi and so forth. Further,electroporation can also be widely used.

The host microorganism into which a target recombinant vector isintroduced can be selected by searching a microorganism thatsimultaneously expresses a drug resistance marker of the vectorcontaining the target DNA and the GDH activity. For example, amicroorganism growing in a selective medium based on the drug resistancemarker and producing GDH can be selected.

As for the culture method of the transformant, culture conditions can beselected by considering nutritional and physiological properties of thehost. In many cases, liquid culture is performed. It is industriallyadvantageous to perform aerobic culture with stirring.

As nutrients of the medium, those usually used for culture ofmicroorganisms can be widely used. As carbon sources, any carboncompounds that can be assimilated can be used, and examples thereofinclude glucose, sucrose, lactose, maltose, lactose, molasses, pyruvicacid and so forth. Further, as nitrogen sources, any nitrogen compoundsthat can be utilized can be used, and examples thereof include peptone,meat extracts, yeast extract, casein hydrolysate, soybean meal alkalineextract and so forth. In addition, phosphoric acid salts, carbonic acidsalts, sulfuric acid salts, salts of magnesium, calcium, potassium,iron, manganese, zinc and so forth, particular amino acids, particularvitamins and so forth are used as required.

Although the culture temperature can be appropriately changed in a rangein which bacteria grow and produce GDH, it is preferably about 20° C. to42° C. The culture time somewhat varies depending on the conditions.However, the culture can be completed at an appropriate time estimatedto give the maximum GDH level, and the culture time is usually about 12to 72 hours. Although pH of the medium can be appropriately changed in arange in which bacteria grow and produce GDH, it is preferably in therange of about pH 6.0 to 9.0.

The culture broth containing cells producing GDH in the culture can becollected and utilized as they are. However, when GDH exists in theculture broth, the culture broth is usually separated into aGDH-containing solution and microorganism cells by filtration,centrifugation or the like in a conventional manner and then used. WhenGDH exists in the cells, the cells are collected from the obtainedculture by means of filtration, centrifugation or the like, and then thecells are disrupted by a mechanical method or an enzymatic method suchas use of lysozyme, and further added with a chelating agent such asEDTA and a surfactant to solubilize GDH, as required, to isolate andcollect GHD as an aqueous solution.

GDH can be precipitated from the GDH-containing solution obtained asdescribed above by, for example, vacuum concentration, membraneconcentration, salting out with ammonium sulfate, sodium sulfate or thelike, or a fractional precipitation with a hydrophilic organic solventsuch as methanol, ethanol and acetone. Further, heat treatment andisoelectric point treatment are also effective purification means. Then,GDH can be purified by a suitable combination of gel filtration using anadsorbent or gel filtration agent, absorption chromatography, ionexchange chromatography and affinity chromatography to obtain purifiedGHD.

A purified enzyme preparation can be obtained by isolation andpurification based on column chromatography. Although the purifiedenzyme preparation is preferably purified to such an extent that asingle band should be obtained in electrophoresis (SDS-PAGE), it maycontain the γ-subunit.

The purified enzyme obtained as described above can be made into powderby, for example, lyophilization, vacuum drying, spray drying or the likeand distributed.

Further, the amino acid sequence of the β-subunit can also be determinedin the same manner as in the determination of the amino acid sequence ofthe α-subunit described later in the examples, and a DNA encoding theβ-subunit can be isolated based on the sequence. Further, the β-subunitcan also be produced by using the obtained DNA. Further, the multimerenzyme can also be produced by using a DNA encoding the α-subunit andDNA encoding the β-subunit.

<4> Glucose Sensor of the Present Invention

The glucose sensor of the present invention is characterized by usingthe enzyme of the present invention (the aforementioned multimer enzymeor peptide enzyme, or the aforementioned multimer enzyme or peptideenzyme containing the γ-subunit), the transformant of the presentinvention, or the microorganism of the present invention (Burkhorderiacepacia KS1 strain) as an enzyme electrode. As the electrode, a carbonelectrode, gold electrode, platinum electrode or the like can be used,and the enzyme of the present invention is immobilized on thiselectrode. Examples of the method for immobilization include a method ofusing a crosslinking reagent, a method of entrapping the enzyme in apolymer matrix, a method of covering the enzyme with a dialysismembrane, methods of using a photocrosslinking polymer, conductivepolymer, oxidation-reduction polymer or the like. Alternatively, theenzyme may be immobilized in a polymer or immobilized on an electrode byadsorption together with an electronic mediator of which typicalexamples are ferrocene and derivatives thereof, or these methods may beused in combination. Typically, the glucose dehydrogenase of the presentinvention is immobilized on a carbon electrode by using glutaraldehyde,and glutaraldehyde is blocked by a treatment with a reagent having anamine group.

The glucose concentration can be measured as follows. A buffer is placedin a constant temperature cell and added with a mediator, and a constanttemperature is maintained. As the mediator, potassium ferricyanide,phenazine methosulfate and for forth can be used. An electrode on whichthe enzyme of the present invention is immobilized is used as a workingelectrode, and a counter electrode (e.g., platinum electrode) and areference electrode (e.g., Ag/AgC electrode) are used. A constantvoltage is applied to the carbon electrode, and after a steady-statecurrent is obtained, a sample containing glucose is added and theincrease of the current is measured. The glucose concentration in thesample can be calculated according to a calibration curve produced byusing glucose solutions having standard concentrations.

<5> Glucose Assay Kit of the Present Invention

The saccharide assay kit of the present invention is characterized byincluding the enzyme of the present invention (the aforementionedmultimer enzyme or peptide enzyme, or the aforementioned multimer enzymeor peptide enzyme containing the γ-subunit). The glucose assay kit ofthe present invention includes the enzyme of the present invention in anamount sufficient for at least one assay. Typically, the kit includes,in addition to the enzyme of the present invention, a buffer, amediator, standard solutions of glucose or the like for creating acalibration curve, which are necessary for the assay, and a guidelinefor use. The enzyme of the present invention can be provided in variousforms, for example, as a lyophilized reagent or a solution in anappropriate storage solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a molecular weight of the enzyme of the present inventiondetermined by native PAGE electrophoresis.

FIG. 2 shows an electrophoretic photograph showing a molecular weight ofthe enzyme of the present invention based on SDS-PAGE electrophoresis.

FIG. 3 shows the optimal reaction temperature (a) and thermal stability(b) of the enzyme of the present invention.

FIG. 4 shows the optimal reaction temperature (a) and thermal stability(b) of a peptide enzyme constituting only the α-subunit of the enzyme ofthe present invention.

FIG. 5 shows results of spectrophotometric analyses of the enzyme of thepresent invention in the absence or presence of glucose before heattreatment (a) and spectrophotometric analyses of the enzyme of thepresent invention in the absence or presence of glucose after heattreatment (b).

FIG. 6 shows responses of a glucose sensor using GDH obtained from atransformant to glucose at various temperatures.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained more specifically with referenceto the following examples.

Example 1 Acquisition of Bacterium Having Glucose DehydrogenaseProducing Ability

The microorganism of the present invention was obtained by collectingsoil near various hot springs in Japan and selecting a bacterium havinga glucose dehydrogenase activity among bacteria utilizing glucose as anutrient from the soil.

The results of investigation of morphological characteristics, growthcharacteristics and physiological characteristics of this strain areshown below.

[Bacteriological characteristics] Gram staining negative Cell morphologyrod-shaped With polar flagellum Mobility positive Number of fragments >5Optimal growth temperature 45° C. Oxidase negative Catalase positiveProduction of acetoin negative Production of H₂S negative Production ofindole negative Acid from glucose positive Arginine dihydrolase negativeUrease negative β-Glucosidase negative Protease negative β-Galactosidasepositive Lysine carboxylase negative Ornithine carboxylase negativeReduction of nitrate positive [Assimilation characteristics] Glycerolpositive Erythritol negative D-Arabinose negative L-Arabinose positiveRibose positive D-Xylose positive L-Xylose negative Adonitol positiveβ-Methyl-xyloside negative Galactose positive D-Glucose positiveD-Fructose positive D-Mannose positive L-Sorbose negative Rhamnosenegative Dulcitol positive Inositol positive Mannitol positive Sorbitolpositive α-Methyl-D-mannoside negative α-Methyl-D-glucoside negativeN-Acetyl-glucosamine positive Amygdaline negative Arbutin negativeEsculin negative Salicin negative Cellobiose negative Maltose negativeLactose negative Melibiose negative Sucrose negative Trehalose positiveInulin negative Melezitose negative D-Raffinose negative Amidon negativeGlycogen negative Xylitol positive β-Gentiobiose negative D-Turanosenegative D-Lyxose negative D-Tagatose negative D-Fucose negativeL-Fucose negative D-Arabitol positive L-Arabitol positive Gluconic acidpositive 2-Ketogluconic acid positive 5-Ketogluconic acid negativeCapric acid positive Adipic acid positive Malic acid positive Citricacid positive Phenyl acetate positive [Oxidation characteristics]Glycerol negative Erythritol negative D-Arabinose negative L-Arabinosepositive Ribose positive D-Xylose positive L-Xylose negative Adonitolpositive β-Methyl-xyloside negative Galactose positive D-Glucosepositive D-Fructose positive D-Mannose positive L-Sorbose negativeRhamnose negative Dulcitol positive Inositol positive Mannitol positiveSorbitol positive α-Methyl-D-mannoside negative α-Methyl-D-glucosidenegative N-Acetyl-glucosamine negative Amygdaline negative Arbutinnegative Esculin positive Salicin negative Cellobiose positive Maltosepositive Lactose positive Melibiose negative Sucrose negative Trehalosepositive Inulin negative Melezitose negative D-Raffinose negative Amidonnegative Glycogen negative Xylitol negative β-Gentiobiose positiveD-Turanose negative D-Lyxose negative D-Tagatose negative D-Fucosepositive L-Fucose negative D-Arabitol positive L-Arabitol positiveGluconic acid negative 2-Ketogluconic acid negative 5-Ketogluconic acidnegative

The taxonomical position of the KS1 strain having the aforementionedbacteriological characteristics was investigated with reference to theBergey's Manual of Determinative Bacteriology, and the strain wasidentified to belong to the genus Burkhorderia, and was a bacterialstrain of Burkhorderia cepacia.

The genus Burkhorderia was conventionally classified into the genusPseudomonas, but is separately classified as the genus Burkhorderia atpresent (Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta, H.,Hashimoto, Y., Ezaki, T. and Arakawa, M., Microbiol. Immunol. Vol. 36(12): 1251-1275 (1992); International Journal of SystematicBacteriology, April, 1993, pp. 398-399).

Further, the inventors of the present invention obtained severalBurkhorderia cepacia strains other than the Burkhorderia cepacia KS1strain, which were deposited at the Institute for Fermentation, Osaka orthe Japan Collection of Microorganisms (JCM), Institute of Physical andChemical Research, and measured glucose dehydrogenase activities of thestrains, and they were confirmed to have the activity. The glucosedehydrogenase activity was measured by the method described later inExample 2. Relative activities of these strains based on the enzymaticactivity of a water-soluble fraction of the KS1 strain, which is takenas 100, are shown in Table 1.

TABLE 1 Glucose dehydrogenase Bacterial activity strain 70° C. 45° C.KS1 Water-soluble 100 100 fraction JCM5506 Water-soluble 100 100fraction Membrane 100 100 fraction JCM5507 Water-soluble 100 100fraction Membrane 100 100 fraction JCM2800 Water-soluble 100 100fraction JCM2801 Water-soluble 100 100 fraction IFO15124 Water-soluble100 100 fraction IFO14595 Water-soluble 100 100 fraction

Example 2 Extraction of Glucose Dehydrogenase

<1> Culture of Cells

As the culture conditions of the bacterium, usual aerobic cultureconditions were used. The cells were cultured at 34° C. for 8 hours in 7L of a medium containing the following ingredients per liter.

Polypeptone 10 g Yeast extract 1 g NaCl 5 g KH₂PO₄ 2 g Glucose 5 g Einol(ABLE Co., Tokyo, Japan) 0.14 g Total volume including distilled water 1L Adjusted pH 7.2

In a volume of 7 L of the culture broth was centrifuged at 9,000×g at 4°C. for 10 minutes to obtain about 60 g of cells.

<2> Preparation of Roughly Purified Fraction

In an amount of 60 g of the cells were dispersed in 10 mM potassiumphosphate buffer (pH 6.0), and a pressure difference of 1,500 Kg/cm² wasapplied to the cells by using a French press (Otake Corporation, Tokyo,Japan) to disrupt cell membranes. The cell extract was centrifuged at8000×g for 10 minutes to remove cellular solid. Further, the supernatantwas subjected to ultracentrifugation at 69,800×g at 4° C. for 90 minutesto obtain about 8 g of a membrane fraction as precipitates.

<3> Purification of Enzyme

The membrane fraction was redispersed in 10 mM potassium phosphatebuffer (pH 6.0) containing 1% of Triton X-100 as a final concentration.Then, the dispersion was slowly stirred overnight at 4° C. After thedispersion was subjected to ultracentrifugation (69,800×g, 4° C., 90minutes), the solubilized membrane fraction was centrifuged again at 4°C. for 15 minutes at 15,000×g to obtain a supernatant.

The solubilized membrane fraction was added with the same volume of 10mM potassium phosphate buffer (pH 8.0) containing 0.2% Triton X-100. Thesolution was dialyzed, and then applied to a DEAE-TOYOPEARL column (22mm ID×20 cm, Tosoh Corporation, Tokyo, Japan) equalized with 10 mMpotassium phosphate buffer (pH 8.0) containing 0.2% Triton X-100.Proteins were eluted with a linear gradient of 0 to 0.15 M NaCl in 10 mMpotassium phosphate buffer (pH 8.0). The flow rate was 5 ml/min. GDH waseluted at a NaCl concentration of about 75 mM. Fractions exhibiting theGDH activity were collected and dialyzed overnight against 10 mMpotassium phosphate buffer (pH 8.0, 4° C.) containing 0.2% Triton X-100.

Further, the dialyzed enzyme solution was applied to a DEAE-5PW column(8.0 mm ID×7.5 cm, Tosoh Corporation, Tokyo, Japan). This column wasequilibrated beforehand with 10 mM potassium phosphate buffer (pH 6.0)containing 0.2% Triton X-100. The proteins were eluted with a lineargradient of 0 to 100 mM NaCl in 10 mM potassium phosphate buffer (pH8.0). The flow rate was 1 ml/min. Fractions exhibiting the GDH activitywere eluted at a NaCl concentration of about 20 mM. The fractions havingthe GDH activity were collected and desalted overnight with 10 mMpotassium phosphate buffer (pH 8.0) containing 0.2% Triton X-100 toobtain the purified enzyme.

The GDH activity was measured according to the following methodthroughout this example and the following examples.

As electron acceptors, 2,6-dichlorophenol-indophenol (DCIP) andphenazine methosulfate (PMS) were used. The reaction was allowed in apolyethylene tube at a predetermined temperature. In a volume of 5 μl ofthe enzyme solution was added to 20 μl of 25 mM Tris-HCl buffer (pH 8.0)containing 0.75 mM PMS and 0.75 mM DCIP. This mixture was left for 1minute beforehand at a constant temperature. The reaction was startedwith the addition of 1 μl of 2 M glucose (final concentration: 77 mM)and left at a constant temperature for 2 minutes. Subsequently, 100 μlof ice-cooled distilled water or 120 μl of 7.5 M urea was added to coolthe sample. A reduction reaction of the electron acceptors due to thedehydrogenation of glucose was monitored by using an ultra-micromeasurement cell (100 μl) and a spectrophotometer (UV160, ShimadzuCorporation, Kyoto, Japan) that enabled measurement using the cell. Thatis, decoloration with time due to the reduction of DCIP was measured at600 nm, which is the absorption wavelength of DCIP. The molar absorbancecoefficient of DCIP (22.23 mM×cm⁻¹) was used. One unit (U) of the enzymewas defined as the amount of oxidizing 1 μM of glucose per minute understandard test conditions. The protein concentration was measured by theLowry method.

Example 3

Native PAGE electrophoresis was performed for the purified enzyme. Theelectrophoresis was performed on 8 to 25% polyacrylamide gradient gelusing a Tris-alanine buffer system containing 1% Triton X-100. The gelwas stained with silver nitrate. As protein markers, thyroglobulin (669kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovineserum albumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25kDa) were used.

Further, activity staining was performed for the native PAGE gel byincubating the gel in the following solution for 30 minutes. At GDHactivity sites, nitroblue tetrazolium was reduced and formazan wasproduced, resulting in development of dark purple color.

200 mM  glucose 0.1 mM  nitroblue tetrazolium 0.3 mM  phenazinemethosulfate 20 mM Tris-HCl buffer (pH 8.0)

From the results of the silver staining in the native PAGE, it wasestimated that the enzyme consisted of a single kind of enzyme and had amolecular weight of about 400 kDa. Further, when the gel was stained forthe activity, the activity was observed at a site of the same mobilityas in the silver staining (See FIG. 1. In the figure, Lane 1 shows theresults of silver staining of marker proteins having standard molecularweights, Lane 2 shows the silver staining of the enzyme, and Lane 4shows the staining for activity of the enzyme). When the enzyme washeated at 70° C. for 30 minutes, the activity unexpectedly remained, andthe enzyme was separated into proteins one of which had the activity andshowed a molecular weight of about 85 kDa (See FIG. 1. In the figure,Lane 3 shows the results of the silver staining of the enzyme heated at70° C. for 30 minutes, and Lane 5 shows the staining for activity of theenzyme heated at 70° C. for 30 minutes). These results suggest that theenzyme consists of subunits.

Example 4

The purified enzyme solution was subjected to SDS-PAGE. SDS-PAGE wasperformed in 8 to 25% gradient polyacrylamide gel by using aTris-tricine buffer. Proteins in the gel were stained with silvernitrate. Separation and development were automatically performed byusing Phast System (Pharmacia) The molecular mass was determined basedon the relative migrations of the standard proteins. The enzyme wasseparated into proteins having molecular weights of about 60 kDa and 43kDa by SDS-PAGE (See FIG. 2. FIG. 2 is an electrophoretic photograph. Inthe figure, Lane 1 shows the results of the silver nitrate staining ofthe marker proteins having standard molecular weights, and Lane 2 showsthe results of the silver nitrate staining of the enzyme). Thus, it wassuggested that the α-subunit of 60 kDa and the β-subunit of 43 kDa werebound in the enzyme, and it was expected that an octamer was formed byfour each of these subunits bonding to each other.

The β-subunit, a protein of 43 kDa separated by SDS-PAGE, wastransferred onto a polyvinylidene fluoride membrane, and then the aminoacid sequence at the N-terminus of the β-subunit was determined by usingan amino acid sequencer (PPSQ-10, Shimadzu Corporation). As a result, itwas found that the amino acid sequence at the N-terminus of the proteinconsisted of 16 residues of the amino acid sequence of SEQ ID NO: 5.

Further, the results obtained with the enzyme subjected to a heattreatment at 70° C. for 30 minutes are shown as Lane 3 in FIG. 2. Basedon this result of SDS-PAGE, it can be estimated that the enzyme waschanged into a single polypeptide having a molecular weight of 60 kDaafter the heat treatment.

Example 5

The enzyme was subjected to gel filtration chromatography. As the gel,TSK Gel G3000SW (Tosoh Corporation) was used, and the gel column (8.0 mmID×30 cm Tosoh Corporation, Tokyo, Japan) was equilibrated with asolution containing 0.3 M NaCl and 0.1% Triton X-100 in 10 mM potassiumphosphate buffer (pH 6.0). Fractions (125 μl) were collected. Sevenkinds of protein markers were used to determine the molecular weight ofthe purified enzyme. As the protein markers, thyroglobulin (669 kDa),ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serumalbumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa)were used.

It was confirmed that the molecular weight of the enzyme was about 380kDa.

Example 6

The optimal temperature of the purified enzyme was examined.

The enzyme was incubated beforehand in Tris-HCl buffer (pH 8.0) at apredetermined temperature for 1 minute, and then the reaction wasstarted. The activity was measured at a predetermined reactiontemperature. The optimal temperature was observed around 45° C. (seeFIG. 3 (a)). Further, a peak was also observed around 75° C., althoughthe activity was lower than the activity around 45° C.

Further, in order to examine thermal stability of the enzyme, the enzymewas left at each constant temperature for 30 minutes, and the residualenzymatic activity was measured at 45° C. (see FIG. 3 (b)).

Example 7

The optimal temperature and the thermal stability of the peptide enzymeconstituting the single oligopeptide having a molecular weight of 60 kDaobtained by heating the enzyme at 70° C. for 30 minutes were examined.

This peptide enzyme showed an optimal temperature higher than that ofthe unheated enzyme as well as thermal stability. There has been noreport about an enzyme having such temperature dependency.

The enzyme was incubated beforehand in Tris-HCl buffer (pH 8.0) at apredetermined temperature for 1 minute, and then the reaction wasstarted. The activity was measured at a predetermined reactiontemperature. The optimal temperature was observed around 75° C. (seeFIG. 4 (a)).

Further, in order to examine thermal stability of the enzyme, the enzymewas left at each constant temperature for 30 minutes, and the residualenzymatic activity was measured at 70° C. (see FIG. 4 (b)).

Example 8

In order to investigate a role of each subunit, spectrophotometricanalysis was performed for GDH before and after the heat treatment.FIGS. 5 (a) and (b) show absorptions of oxidized and reduced GDHs beforeand after heat treatment (in the presence of glucose). The absorptionwavelength of the oxidized GDH before heat treatment, which was theoriginal GDH, showed a characteristic peak at 409 nm. Further, the peakshifted to 417 nm in the presence of glucose, and two more peaks wereobserved at 523 nm and 550 nm (FIG. 5 (a)). In contrast, the GDH afterthe heat treatment no longer showed the characteristic peak at 409 nm(FIG. 5 (b)), and no significant difference was observed between theoxidized and reduced GDHs.

The absorption wavelength of the oxidized GDH before heat treatment,which was the original GDH, was similar to the absorption wavelength ofalcohol dehydrogenase or aldehyde dehydrogenase comprising adehydrogenase cytochrome complex of Gluconobacter sp. or Acetobacter sp.(refer to the following references: Adachi, O., Tayama, K., Shinagawa,E., Matsushita, K. and Ameyama, M., Agr. Biol. Chem., 42, 2045-2056(1978); Adachi, O., Miyagawa, E., Matsushita, K. and Ameyama, M., Agr.Biol. Chem., 42, 2331-2340 (1978); Ameyama, M. and Adachi, O., MethodsEnzymol., 89, 450-457 (1982); Adachi, O., Tayama, K., Shinagawa, E.,Matsushita, K. and Ameyama, M., Agr. Biol. Chem., 44, 503-515 (1980);Ameyama, M. and Adachi, O., Methods Enzymol., 89, 491-497 (1982)).

The results indicated a possibility that the oligomer complex of the GDHcontained cytochrome. Therefore, it can be considered that the observedwavelength similar to that of cytochrome C is attributable to theβ-subunit and was lost during the heat treatment, and thus the β-subunitconsists of cytochrome C.

Example 9

A band containing the β-subunit obtained by the electrophoresis inExample 4 was excised, and the amino acid sequence was analyzed by usinga peptide sequencer (PPSQ-10, Shimadzu Corporation). As a result, theN-terminus amino acid sequence consisting of 16 residues shown in SEQ IDNO: 5 could be obtained.

It was attempted to amplify a gene region encoding the aforementionedN-terminus amino acid sequence of 16 residues by PCR based on thepeptide sequence. That is, two of PCR primers were designed, which had anucleotide sequence on the forward side (SEQ ID NO: 6) corresponding to5 residues at the N-terminus and a nucleotide sequence on the reverseside (SEQ ID NO: 7) corresponding to the antisense strand of 5 residuesat the C-terminus in the peptide chain of the 16 residues. When PCR wasperformed in a conventional manner for the KS1 strain genome by usingthis pair of PCR primers, a gene fragment of about 50 bp was amplified.When the nucleotide sequence of this gene fragment was determined in aconventional manner, a nucleotide sequence of 58 nucleotides containingthe pair of PCR primers were decoded. Among these nucleotides, 18nucleotides excluding the PCR primers were analyzed, and a gene sequencecorresponding to a region from Pro, which was the 6th residue from theN-terminus side of the aforementioned 16 residues at the N-terminus ofthe β-subunit, to Arg, which was the 11th residue, was found (SEQ ID NO:8). Thus, it was found that the amplified gene fragment included thegene fragment of the 1-subunit.

Further, it was also found that the β-subunit existed after 22 aminoacid residues following the β-subunit. This was based on a finding that,since the amino acid sequence at the N-terminus of the purified subunitdetermined in Example 4 matched 5 amino acid residues translated fromthe nucleotide sequence of the nucleotide numbers 2452 to 2466 in SEQ IDNO: 1, these sequences are identical.

Furthermore, it is inferred that the nucleotide sequence of thenucleotide numbers 2386 to 2451 in SEQ ID NO: 1 is the signal peptide ofthe β-subunit. The amino acid sequence encoded by this nucleotidesequence corresponds to the amino acid numbers 1 to 22 in the amino acidsequence of SEQ ID NO: 4.

Example 10

The purified enzyme and a commercially available NAD coenzyme GDH(abbreviated as “NAD-GDH”) were added and mixed in 50 mM potassiumphosphate buffer (pH 7.5) containing 0.1% Triton X-100 and 1 mM CaCl₂ ata concentration of 100 U/L each. Each solution was placed in a hot tankat 60° C., and the residual activity was measured.

TABLE 2 Residual relative activity (%) Time (min) NAD-GDH The enzyme GDH0 100 100 15 20 100 30 5 100

It was confirmed that the enzyme had surprising thermal stability incomparison with that of the currently commercially available GDH enzyme.It was found that the enzyme was a novel enzyme that is totallydifferent from the commercially available NAD-GDH.

Example 10 Isolation of Gene Encoding α-Subunit of GDH

<1> Preparation of Chromosomal DNA from Burkhorderia cepacia KS1 Strain

A chromosomal gene was prepared from the Burkhorderia cepacia KS1 strainin a conventional manner. That is, the bacterial strain was shakenovernight at 34° C. by using a TL liquid medium (10 g of polypeptone, 1g of yeast extract, 5 g of NaCl, 2 g of KH₂PO₄, 5 g of glucose in 1 L,pH 7.2). The grown cells were collected by using a centrifugal machine.The cells were suspended in a solution containing 10 mM NaCl, 20 mMTris-HCl (pH 8.0), 1 mM EDTA, 0.5% SDS and 100 μg/ml proteinase K andtreated at 50° C. for 6 hours. This mixture was added with an equivalentvolume of phenol-chloroform and stirred at room temperature for 10minutes, and then the supernatant was collected by using a centrifugalmachine. The supernatant was added with sodium acetate at a finalconcentration of 0.3 M and overlaid with two-fold volume of ethanol toprecipitate chromosomal DNA in the intermediate layer. The DNA was takenup with a glass rod, washed with 70% ethanol and dissolved in anappropriate amount of TE buffer to obtain a chromosomal DNA solution.

<2> Determination of N-Terminus Amino Acid Sequence of α-Subunit of GDH

GDH purified in the same manner as in Example 2 was concentrated bylyophilization and developed by SDS-electrophoresis using 12.5%polyacrylamide to isolate the α-subunit. The α-subunit thus obtained wastransferred onto a polyvinylidene fluoride membrane, and then theN-terminus amino acid sequence was determined by using an amino acidsequencer (PPSQ-10, Shimadzu Corporation). As a result, it was foundthat the enzyme included a peptide sequence consisting of 11 residues ofthe amino acid numbers 2 to 12 in the amino acid sequence of SEQ ID NO:3.

<3> Cloning of Gene Encoding α-Subunit

In an amount of 1 μg of the DNA prepared in <1> was subjected to limiteddigestion with a restriction enzyme Sau3AI and treated with calfintestinal alkaline phosphatase (CIAP). Separately, SuperCosI (obtainedfrom STRATAGENE), which is a cosmid, was treated with BamHI, and the DNAfragment obtained by the limited digestion of the chromosomal DNAfragment derived from the α-15 strain with Sau3AI was incorporated intoSuperCosI by using T4 DNA ligase. Escherichia coli XL-1 Blue MR(obtained from STRATAGENE) was transformed with the obtained recombinantDNA. Transformants were selected on an LB agar medium containing 10μg/ml neomycin and 25 μg/ml ampicillin based on neomycin resistance andampicillin resistance, which are antibiotic resistances of SuperCosI.The obtained transformants were cultured in the LB liquid medium. Thesetransformant cells were collected and suspended in a reagent formeasuring the GDH activity, and clones were selected by usingdehydrogenase activity for glucose as an index. As a result, one clonestrain showing the glucose dehydrogenase activity was obtained.

<4> Subcloning

DNA fragments containing the target gene were prepared from the cosmid,SuperCosI, containing the gene encoding the α-subunit obtained in <3>.The inserted gene fragments were excised from the cosmid by using arestriction enzyme NotI. These DNA fragments were treated with arestriction enzyme XbaI and incorporated into plasmid pUC18 digestedwith XbaI. The Escherichia coli DH5αMCR strain was transformed with theplasmid pUC18 containing each insert fragment, and colonies grown on anLB agar medium containing 50 μg/ml ampicillin were collected. Theobtained transformants were cultured in a liquid LB medium and examinedfor the GDH activity in the cells in the same manner as in <3>. As aresult, a strain showing the GDH activity was obtained from onetransformant. The plasmid was extracted from this transformant, and theinserted DNA fragment was analyzed. As a result, an insert fragment ofabout 8.8 kbp was confirmed. This plasmid was designated as pKS1.

<5> Determination of Nucleotide Sequence

The nucleotide sequence of the inserted DNA fragment in pKS1 wasdetermined according to the restriction enzyme analysis and aconventional method. As a result, the sequence of the DNA encoding theN-terminus amino acid sequence of the α-subunit found in <2> wasconfirmed in this inserted DNA fragment, and an open reading framecontaining this sequence was found. The determined nucleotide sequenceand the amino acid sequence that can be encoded by this nucleotidesequence are as shown in SEQ ID NOS: 1 and 3. The molecular weight of aprotein obtained from the amino acid sequence was 59,831 Da andsubstantially matched the molecular weight of 60 kDa obtained bySDS-PAGE of the α-subunit of the Burkhorderia cepacia KS1 strain.

Since the nucleotide sequence of the α-subunit was determined, a vectorwas produced by using the aforementioned structural gene of theα-subunit, and a transformant was further produced with this vector.

First, a gene to be inserted into the vector was prepared as follows.

Amplification was performed by PCR using a genome fragment derived fromthe KS1 strain as a template so that a desired restriction enzyme siteshould be included. The following pair of oligonucleotide primers wereused in PCR.

(Forward) 5′-CCCAAGCTTGGGCCGATACCGATACGCA-3′ (SEQ ID NO: 9) (Reverse)5′-GAGAAGCTTTCCGCACGGTCAGACTTCC-3′ (SEQ ID NO: 10)

The gene amplified by PCR was digested with a restriction enzyme HindIIIand inserted into the expression vector pFLAG-CTS (SIGMA) at its cloningsite, HindIII site. The obtained plasmid was designated as pFLAG-CTS/α.

The Escherichia coli DH5αMCR strain was transformed with theaforementioned plasmid pFLAG-CTS/α, and a colony grown on an LB agarmedium containing 50 μg/ml ampicillin was collected.

Further, when the open reading frame of the pKS1 insert fragment wassearched in the upstream of the α-subunit, a structural gene of 507nucleotides encoding a polypeptide comprising 168 amino acid residuesshown in SEQ ID NO: 2 (nucleotide numbers 258 to 761 in SEQ ID NO: 1)was newly found. This structural gene was considered to encode theγ-subunit.

Since it was found that the region encoding the γ-subunit existedupstream from the coding region of the α-subunit, a recombinant vectorcontaining a gene having a polycistronic structure continuouslyincluding the γ-subunit and the α-subunit was produced, and atransformant introduced with this vector was constructed.

First, a gene to be inserted into the vector was prepared as follows.

Amplification was performed by PCR using a genome fragment of the KS1strain continuously including the structural gene of the γ-subunit andthe structural gene of the α-subunit as a template so that a desiredrestriction enzyme site should be included. The following pair ofoligonucleotide primers were used for PCR.

(Forward) (SEQ ID NO: 11) 5′-CATGCCATGGCACACAACGACAACACT-3′ (Reverse)(SEQ ID NO: 12) 5′-CCCAAGCTTGGGTCAGACTTCCTTCTTCAGC-3′

The 5′ end and the 3′ end of the gene amplified by PCR were digestedwith NcoI and HindIII, respectively, and the gene was inserted into thevector pTrc99A (Pharmacia) at its cloning site, NcoI/HindIII site. Theobtained plasmid was designated as pTrc99A/γ+α.

The Escherichia coli DH5αMCR strain was transformed with theaforementioned plasmid pTrc99A/γ+α, and a colony grown on an LB agarmedium containing 50 μg/ml ampicillin was collected.

Example 11 Production of α-Subunit of GDH by Recombinant Escherichiacoli

The α-subunit was produced by using the Escherichia coli DH5αMCR straintransformed with each of the aforementioned plasmids pKS1, pFLAG-CTS/αand pTrc99A/γ+α. Each transformant was inoculated into 3 ml of LB mediumcontaining 50 μg/ml ampicillin and cultured at 37° C. for 12 hours, andcells were collected by using a centrifugal machine. The cells weredisrupted by using a French press (1500 kgf), and a membrane fraction(10 mM potassium phosphate buffer, pH 6.0) was isolated byultracentrifugation (160,400×g, 4° C., 90 minutes).

Example 12 Assay of Glucose

First, the GDH activity in each of the aforementioned membrane fractionswas confirmed. Specifically, visual determination was performed by usinga 10 mM potassium phosphate buffer (pH 7.0) containing 594 μMmethylphenazine methosulfate (mPMS) and 5.94 μM2,6-dichlorophenol-indopheol (DCIP). The results are shown below. Thenumber of + represents the degree of color change from blue tocolorless.

-   -   Membrane fraction of cultured transformant transformed with        pFLAG-CTS/α: +    -   Membrane fraction of cultured transformant transformed with        pKS1: ++    -   Membrane fraction of cultured transformant transformed with        pTrc99A/γ+α:

The GDH activity of the membrane fraction of the cultured transformanttransformed with pFLAG-CTS/α incorporated only with the α-subunit wasthe lowest, and the GDH activity of the membrane fraction of thecultured transformant transformed with pTrc99A/γ+α, with which a vectorwas efficiently constructed, was the highest.

Although the α-subunit was expressed even in the transformanttransformed with a vector using only the structural gene of theα-subunit, the α-subunit could be efficiently obtained by using a vectorcontaining the structural gene of the γ-subunit and the structural geneof the α-subunit in combination.

Glucose was assayed by using the glucose dehydrogenase of the presentinvention. The enzymatic activity of the glucose dehydrogenaseα-subunit) of the present invention was measured by using glucose atvarious concentrations. The GDH activity was measured in 10 mM potassiumphosphate buffer (pH 7.0) containing 594 μM methylphenazine methosulfate(mPMS) and 5.94 μM 2,6-dichlorophenol-indopheol (DCIP). An enzyme sampleand glucose as a substrate were added and incubated at 37° C., andchange in the absorbance of DCIP at 600 nm was monitored by using aspectrophotometer. The absorbance decreasing rate was measured as anenzymatic reaction rate. Glucose could be quantified in the range of0.01 to 1.0 mM by using the GDH of the present invention.

Example 13 Preparation and Evaluation of Glucose Sensor

The glucose dehydrogenase (25 units) of the present invention obtainedin Example 2 was added with 20 mg of carbon paste and lyophilized. Thesewere sufficiently mixed, applied only on a surface of a carbon pasteelectrode already filled with about 40 mg of carbon paste and polishedon a filter paper. This electrode was treated in 10 mM MOPS buffer (pH7.0) containing 1% glutaraldehyde at room temperature for 30 minutes andthen treated in 10 mM MOPS buffer (pH 7.0) containing 20 mM lysine atroom temperature for 20 minutes to block glutaraldehyde. This electrodewas equilibrated in 10 mM MOPS buffer (pH 7.0) at room temperature for 1hour or longer. The electrode was stored at 4° C.

By using the aforementioned electrode as a working electrode, an Ag/AgClelectrode as a reference electrode and a Pt electrode as a counterelectrode, a response current value was measured upon addition ofglucose. The 10 mM potassium phosphate buffer containing 1 mMmethoxy-PMS was used as the reaction solution, and a potential of 100 mVwas applied for the measurement.

Glucose concentration was measured by using the produced enzyme sensor.Glucose could be quantified in the range of 0.05 to 5.0 mM by using theenzyme sensor on which the glucose dehydrogenase of the presentinvention was immobilized (FIG. 6).

Example 14 Preparation and Evaluation of Glucose Sensor by GDH Obtainedfrom Transformant

In an amount of 10 U of the α-subunit (249 U/mg protein) of the presentinvention obtained in Example 12 was added with 50 mg of carbon pasteand lyophilized. These were sufficiently mixed, applied only on asurface of a carbon paste electrode already filled with about 40 mg ofcarbon paste and polished on a filter paper. This electrode was treatedin a 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde at roomtemperature for 30 minutes and then treated in 10 mM MOPS buffer (pH7.0) containing 20 mM lysine at room temperature for 20 minutes to blockglutaraldehyde. This electrode was equilibrated in 10 mM MOPS buffer (pH7.0) at room temperature for 1 hour or longer. The electrode was storedat 4° C.

By using the aforementioned electrode as a working electrode, an Ag/AgClelectrode as a reference electrode and a Pt electrode as a counterelectrode, a response current value was measured upon addition ofglucose. The 10 mM potassium phosphate buffer containing 1 mMmethoxy-PMS was used as the reaction solution, and the measurement wasperformed for glucose aqueous solutions of various concentrations at 25°C. and 40° C. with applying a potential of 100 mV.

It was confirmed that, when the glucose concentration was measured byusing the produced enzyme sensor, a current corresponding to eachconcentration was obtained.

INDUSTRIAL APPLICABILITY

According to the present invention, an enzyme that has high substratespecificity, can be produced at a low cost and is not affected by oxygendissolved in a measurement sample, in particular, novel glucosedehydrogenase having superior thermal stability, and a method forproducing the enzyme could be provided. Further, a novel bacterialstrain of Burkhorderia cepacia producing the enzyme was obtained. Aglucose sensor effective for measurement of glucose can also be providedby using an enzyme electrode containing the enzyme or the bacterialstrain.

Further, since the glucose dehydrogenase gene, a peptide that enablesefficient expression of the gene and a DNA encoding this peptide werefound by the present invention, a large amount of GDH can be prepared byusing recombinant DNA techniques based on the gene.

1. An isolated protein having the amino acid sequence of SEQ ID NO: 2.